3C-like protease

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DOI.10.1126.science.abb4489.S2.png
SARS-CoV-2 main proteinase dimer with the catalytic dyad (H41; C145) in complex with a covalent peptidomimetic protease inhibitor ("11a", magenta). From PDB: 6LZE ​. [1]
Identifiers
EC no. 3.4.22.69
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Search
PMC articles
PubMed articles
NCBI proteins
Peptidase C30, Coronavirus endopeptidase
Identifiers
SymbolPeptidase_C30
Pfam PF05409
InterPro IPR008740
PROSITE PS51442
MEROPS C30
SCOP2 d1q2wb1 / SCOPe / SUPFAM
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

The 3C-like protease (3CLpro) or main protease (Mpro), formally known as C30 endopeptidase or 3-chymotrypsin-like protease, [2] is the main protease found in coronaviruses. It cleaves the coronavirus polyprotein at eleven conserved sites. It is a cysteine protease and a member of the PA clan of proteases. It has a cysteine-histidine catalytic dyad at its active site and cleaves a Gln–(Ser/Ala/Gly) peptide bond.

Contents

The Enzyme Commission refers to this family as SARS coronavirus main proteinase (Mpro; EC 3.4.22.69). The 3CL protease corresponds to coronavirus nonstructural protein 5 (nsp5). The "3C" in the common name refers to the 3C protease (3Cpro) which is a homologous protease found in picornaviruses.

Function

The 3C-like protease is able to catalytically cleave a peptide bond between a glutamine at position P1 and a small amino acid (serine, alanine, or glycine) at position P1'. The SARS coronavirus 3CLpro can for instance self-cleave the following peptides: [3] [4] [5]

TSAVLQ-SGFRK-NH2 and SGVTFQ-GKFKK are the two peptides corresponding to the two self-cleavage sites of the SARS 3C-like proteinase

The protease is important in the processing of the coronavirus replicase polyprotein ( P0C6U8 ). It is the main protease in coronaviruses and corresponds to nonstructural protein 5 (nsp5). [6] It cleaves the coronavirus polyprotein at 11 conserved sites. The 3CL protease has a cysteine-histidine catalytic dyad at its active site. [4] The sulfur of the cysteine acts as a nucleophile and the imidazole ring of the histidine as a general base. [7]

Substrate preferences for 3CL proteases (from table 2) [8]
PositionSubstrate preference
P5No strong preference
P4Small hydrophobic residues
P3Positively charged residue
P2High hydrophobicity and absence of beta-branch
P1 Glutamine
P1'Small residues
P2'Small residues
P3'No strong preference

Nomenclature

Alternative names provided by the EC include 3CLpro, 3C-like protease, coronavirus 3C-like protease, Mpro, SARS 3C-like protease, SARS coronavirus 3CL protease, SARS coronavirus main peptidase, SARS coronavirus main protease, SARS-CoV 3CLpro enzyme, SARS-CoV main protease, SARS-CoV Mpro and severe acute respiratory syndrome coronavirus main protease.

As a treatment target

Nirmatrelvir bound to 3CL PDB: 7RFW Nirtalmatrelvir on 3CL.png
Nirmatrelvir bound to 3CL PDB: 7RFW ​
Nirmatrelvir, a 3CLpro inhibitor developed by Pfizer in phase II/III clinical trials as a combination drug with ritonavir. PF-07321332.svg
Nirmatrelvir, a 3CLpro inhibitor developed by Pfizer in phase II/III clinical trials as a combination drug with ritonavir.

A number of protease inhibitors are being developed targeting 3CLpro and homologous 3Cpro, including CLpro-1, GC376, rupintrivir, lufotrelvir, PF-07321332, and AG7404. [11] [12] [13] [14] [1] The intravenous administered prodrug PF-07304814 (lufotrelvir) entered clinical trials in September 2020. [15]

After clinical trials, in December 2021, the oral medication nirmatrelvir (formerly PF-07321332) became commercially available under emergency use authorizations (EUA), as part of the nirmatrelvir/ritonavir combination therapy (brand name Paxlovid). [16] [17] In May 2023, the medication got full FDA approval for high-risk adults, while children 12–18 were still covered under the EUA. [18]

The 3C-like protease inhibitor ensitrelvir received authorization to treat COVID-19 in Japan in 2022. [19] [20]

In 2022, an ultralarge virtual screening campaign of 235 million molecules was able to identify a novel broad-spectrum inhibitor targeting the main protease of several coronaviruses. It is unusually not a peptidomimetic. [21]

A ligand-binding diagram showing the amino acid residues in contact with a covalently bound peptidomimetic protease inhibitor. The small red spheres are water molecules. Doi.10.1126.science.abb4489.F3.large.C.jpg
A ligand-binding diagram showing the amino acid residues in contact with a covalently bound peptidomimetic protease inhibitor. The small red spheres are water molecules.

Other 3C(-like) proteases

3C-like proteases (3C(L)pro) are widely found in (+)ssRNA viruses. All of them are cysteine proteases with a chymotrypsin-like fold (PA clan), using a catalytic dyad or triad. They share some general similarities on substrate specificity and inhibitor effectiveness. They are divided into subfamilies by sequence similarity, corresponding to the family of viruses they are found in: [22]

Additional members are known from Potyviridae and non-Coronaviridae Nidovirales . [23]

See also

Related Research Articles

<span class="mw-page-title-main">SARS-related coronavirus</span> Species of coronavirus causing SARS and COVID-19

Betacoronavirus pandemicum is a species of virus consisting of many known strains. Two strains of the virus have caused outbreaks of severe respiratory diseases in humans: severe acute respiratory syndrome coronavirus 1, the cause of the 2002–2004 outbreak of severe acute respiratory syndrome (SARS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the pandemic of COVID-19. There are hundreds of other strains of SARSr-CoV, which are only known to infect non-human mammal species: bats are a major reservoir of many strains of SARSr-CoV; several strains have been identified in Himalayan palm civets, which were likely ancestors of SARS-CoV-1.

Protease inhibitors (PIs) are medications that act by interfering with enzymes that cleave proteins. Some of the most well known are antiviral drugs widely used to treat HIV/AIDS, hepatitis C and COVID-19. These protease inhibitors prevent viral replication by selectively binding to viral proteases and blocking proteolytic cleavage of protein precursors that are necessary for the production of infectious viral particles.

<span class="mw-page-title-main">Ritonavir</span> Antiretroviral medication

Ritonavir, sold under the brand name Norvir, is an antiretroviral medication used along with other medications to treat HIV/AIDS. This combination treatment is known as highly active antiretroviral therapy (HAART). Ritonavir is a protease inhibitor, though it now mainly serves to boost the potency of other protease inhibitors. It may also be used in combination with other medications to treat hepatitis C and COVID-19. It is taken by mouth.

<span class="mw-page-title-main">Picornain 3C</span>

Picornain 3C is a protease found in picornaviruses, which cleaves peptide bonds of non-terminal sequences. Picornain 3C’s endopeptidase activity is primarily responsible for the catalytic process of selectively cleaving Gln-Gly bonds in the polyprotein of poliovirus and with substitution of Glu for Gln, and Ser or Thr for Gly in other picornaviruses. Picornain 3C are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Picornain 3C is encoded by enteroviruses, rhinoviruses, aphtoviruses and cardioviruses. These genera of picoviruses cause a wide range of infections in humans and mammals.

<span class="mw-page-title-main">Rupintrivir</span> Chemical compound

Rupintrivir is a peptidomimetic antiviral drug which acts as a 3C and 3CL protease inhibitor. It was developed for the treatment of rhinoviruses, and has subsequently been investigated for the treatment of other viral diseases including those caused by picornaviruses, norovirus, and coronaviruses, such as SARS and COVID-19.

<span class="mw-page-title-main">3CLpro-1</span> Chemical compound

3CLpro-1 is an antiviral drug related to rupintrivir which acts as a 3CL protease inhibitor and was originally developed for the treatment of human enterovirus 71. It is one of the most potent of a large series of compounds developed as inhibitors of the viral enzyme 3CL protease, with an in vitroIC50 of 200 nM. It also shows activity against coronavirus diseases such as SARS and MERS, and is under investigation as a potential treatment agent for the viral disease COVID-19.

<span class="mw-page-title-main">GC376</span> Broad-spectrum antiviral medication

GC376 is a broad-spectrum antiviral medication under development by the biopharmaceutical company Anivive Lifesciences for therapeutic uses in humans and animals. Anivive licensed the exclusive worldwide patent rights to GC376 from Kansas State University. As of 2020, GC376 is being investigated as a treatment for COVID-19. GC376 shows activity against many human and animal viruses, including coronavirus and norovirus; the most extensive research has been multiple in vivo studies in cats treating a coronavirus, which causes deadly feline infectious peritonitis. Other research supports use in porcine epidemic diarrhea virus.

<span class="mw-page-title-main">Aloxistatin</span> Chemical compound

Aloxistatin is a drug which acts as a cysteine protease inhibitor and has anticoagulant effects. It is a synthetic analogue of E-64, a natural product derived from fungi. It was researched for the treatment of muscular dystrophy but was not successful in human clinical trials, though it has continued to be investigated for treatment of spinal cord injury, stroke and Alzheimer's disease.

<span class="mw-page-title-main">Nirmatrelvir</span> COVID-19 antiviral medication

Nirmatrelvir is an antiviral medication developed by Pfizer which acts as an orally active 3C-like protease inhibitor. It is part of a nirmatrelvir/ritonavir combination used to treat COVID-19 and sold under the brand name Paxlovid.

<span class="mw-page-title-main">GRL-0617</span> Chemical compound

GRL-0617 is a drug which is one of the first compounds discovered that acts as a selective small-molecule inhibitor of the protease enzyme papain-like protease (PLpro) found in some human pathogenic viruses, including the coronavirus SARS-CoV-2. It has been shown to inhibit viral replication in silico and in vitro.

<span class="mw-page-title-main">Lufotrelvir</span> Chemical compound

Lufotrelvir (PF-07304814) is an antiviral drug developed by Pfizer which acts as a 3CL protease inhibitor. It is a prodrug with the phosphate group being cleaved in vivo to yield the active agent PF-00835231. Lufotrelvir is in human clinical trials for the treatment of COVID-19, and shows good activity against COVID-19 including several variant strains, but unlike the related drug nirmatrelvir it is not orally active and must be administered by intravenous infusion, and so has been the less favoured candidate for clinical development overall.

The COVID Moonshot is a collaborative open-science project started in March 2020 with the goal of developing an un-patented oral antiviral drug to treat SARS-CoV-2, the virus causing COVID-19. COVID Moonshot researchers are targeting the proteins needed to form functioning new viral proteins. They are particularly interested in proteases such as 3C-like protease (Mpro), a coronavirus nonstructural protein that mediates the breaking and replication of proteins.

ORF1ab refers collectively to two open reading frames (ORFs), ORF1a and ORF1b, that are conserved in the genomes of nidoviruses, a group of viruses that includes coronaviruses. The genes express large polyproteins that undergo proteolysis to form several nonstructural proteins with various functions in the viral life cycle, including proteases and the components of the replicase-transcriptase complex (RTC). Together the two ORFs are sometimes referred to as the replicase gene. They are related by a programmed ribosomal frameshift that allows the ribosome to continue translating past the stop codon at the end of ORF1a, in a -1 reading frame. The resulting polyproteins are known as pp1a and pp1ab.

<span class="mw-page-title-main">Nidoviral papain-like protease</span> Papain-like protease protein domain

The nidoviral papain-like protease is a papain-like protease protein domain encoded in the genomes of nidoviruses. It is expressed as part of a large polyprotein from the ORF1a gene and has cysteine protease enzymatic activity responsible for proteolytic cleavage of some of the N-terminal viral nonstructural proteins within the polyprotein. A second protease also encoded by ORF1a, called the 3C-like protease or main protease, is responsible for the majority of further cleavages. Coronaviruses have one or two papain-like protease domains; in SARS-CoV and SARS-CoV-2, one PLPro domain is located in coronavirus nonstructural protein 3 (nsp3). Arteriviruses have two to three PLP domains. In addition to their protease activity, PLP domains function as deubiquitinating enzymes (DUBs) that can cleave the isopeptide bond found in ubiquitin chains. They are also "deISGylating" enzymes that remove the ubiquitin-like domain interferon-stimulated gene 15 (ISG15) from cellular proteins. These activities are likely responsible for antagonizing the activity of the host innate immune system. Because they are essential for viral replication, papain-like protease domains are considered drug targets for the development of antiviral drugs against human pathogens such as MERS-CoV, SARS-CoV, and SARS-CoV-2.

<span class="mw-page-title-main">Nirmatrelvir/ritonavir</span> Antiviral combination medication

Nirmatrelvir/ritonavir, sold under the brand name Paxlovid, is a co-packaged medication used as a treatment for COVID‑19. It contains the antiviral medications nirmatrelvir and ritonavir and was developed by Pfizer. Nirmatrelvir inhibits SARS-CoV-2 main protease, while ritonavir is a strong CYP3A inhibitor, slowing down nirmatrelvir metabolism and therefore boosting its effect. It is taken by mouth.

<span class="mw-page-title-main">Papain-like protease</span> Protein family of cysteine protease enzymes

Papain-like proteases are a large protein family of cysteine protease enzymes that share structural and enzymatic properties with the group's namesake member, papain. They are found in all domains of life. In animals, the group is often known as cysteine cathepsins or, in older literature, lysosomal peptidases. In the MEROPS protease enzyme classification system, papain-like proteases form Clan CA. Papain-like proteases share a common catalytic dyad active site featuring a cysteine amino acid residue that acts as a nucleophile.

<span class="mw-page-title-main">Olgotrelvir</span> COVID-19 SARS-CoV-2 3CL-protease-inhibitor antiviral drug

Olgotrelvir (STI-1558) is an experimental antiviral medication being studied as a potential treatment for COVID-19. It is believed to work by inhibiting the SARS-CoV-2 main protease (Mpro), a key enzyme that SARS-CoV-2 needs to replicate, and by blocking viral entry.

<span class="mw-page-title-main">Jun12682</span> Antiviral drug

Jun12682 is an experimental antiviral medication being studied as a potential treatment for COVID-19. It is believed to work by inhibiting SARS-CoV-2 papain-like protease (PLpro), a crucial enzyme for viral replication.

<span class="mw-page-title-main">MK-7845</span> Antiviral drug

MK-7845 is an experimental antiviral medication being studied as a potential treatment for COVID-19. It is believed to work by inhibiting SARS-CoV-2 main protease (3CLpro), a crucial enzyme for viral replication.

<span class="mw-page-title-main">Ibuzatrelvir</span> Antiviral drug

Ibuzatrelvir is an experimental antiviral drug being developed by Pfizer for the treatment of COVID-19. It is a second-generation improvement over nirmatrelvir which has a similar chemical structure. One of the disadvantages of nirmatrelvir is that it has low metabolic stability and must be given in combination with ritonavir to limit its metabolic degradation in the body. Ibuzatrelvir incorporates modifications to the chemical structure of nirmatrelvir that give it enhanced oral bioavailability, so it does not require coadministration with ritonavir.

References

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  19. ↑ "Xocova (Ensitrelvir Fumaric Acid) Tablets 125mg Approved in Japan for the Treatment of SARS-CoV-2 Infection, under the Emergency Regulatory Approval System". Shionogi (Press release). 22 November 2022. Retrieved 28 November 2022.
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